The freshwater flatworm
Although the Alps are a relatively young mountain range (roughly 80 million
years old; Veit, 2002), numerous geological and climatic events have
repeatedly influenced the geographical structure of these mountains (Willet
et al., 2006), with strong impacts on diversity and the distribution of
Alpine life (Thienemann, 1950; Holdhaus, 1954; de Latin, 1967;
Schönswetter et al., 2005). In this study we analyzed molecular sequence
divergence of the freshwater flatworm
Map of the Alps with locations of sampled
In August 2006 and May 2007 10–20 individuals of
The DNeasy™ Tissue Kit (Qiagen) was used to extract DNA following the
protocol provided for animal tissues. For the mitochondrial cytochrome
oxidase subunit I (CO I) gene the primers Pr-a2 (5'-AGC TGC AGT TTT GGT TTT
TTG GAC ATC CTG AGG T-3'; Bessho et al., 1992) and CO13B (5'-AAG TGT TGN GGR
AAR AAN GT-3'; Telford et al., 2000) were used for amplification. These
primers increased the length of the sequenced fragment considered in our
previous study (see Brändle et al., 2007) from 382 to 555 bp. 18S
rRNA gene fragments with a length of 544 bp were amplified using the primers
18S1 (5'-TAC TGT TGA TCC TGC CAG TA-3'; Kuznedelov et al., 1996) and 18S2
(5'- ATT ACC GCG GCT GCT GGC ACC-3'; Kuznedelov et al., 1996). The fragments
were amplified in a thermocycler (Eppendorf Mastercycler, Hamburg, Germany).
The PCR conditions for the COI gene consisted of 5 min initial denaturation
at 95
Partial COI gene and 18S rRNA gene sequences were aligned using BioEdit
7.0.5.3 (Hall, 1999). In a number of sequences particularly within the COI
fragment, some positions showed ambiguities and were discarded. These
exclusions reduced the length of the COI gene fragment to 450 bp and those
of the 18S RNA gene fragment to 531 bp. Thus, the total length of the
combined fragments used for phylogenetic inference was 981 bp. We also
translated the COI gene sequences into amino acid sequences according to the
flatworm mitochondrial genetic code (Nei and Gojobori, 1986) using MEGA 5.05
(Tamura et al., 2011). Except for one position in the sequence of
The number of substitutions and genetic distances between haplotypes of both
fragments and the combined data sets were calculated with MEGA 5.05. Spatial
structuring of the sampled populations was analyzed by means of correlation
analysis of pairwise genetic versus pairwise geographic distances among the sample
sites from the Alps (all 37 sampling sites) using patristic distances
inferred from the Bayesian tree of the COI gene fragment (see below).
Patristic distances were calculated with the program PATRISTIC (Fourment and
Gibbs, 2006). We tested this matrix correlation using a Mantel test as
implemented in the “vegan” package (Oksanen et al., 2013) in the software
R (
Phylogenetic relationships among haplotypes were analyzed with MRBAYES
version 3.1.2 (Ronquist and Huelsenbeck, 2003). To select an appropriate
evolutionary model of nucleotide substitution for each nucleotide fragment,
the approach of Minin et al. (2003) as implemented in the Perl script DT-ModSel
was used. In contrast to the likelihood-ratio tests implemented in
MrModeltest 2.2 (Nylander, 2004), this approach of performance-based model
selection uses the Bayesian information criterion and considers the relative
branch-length error as a performance measure in a consistent framework with
penalty for overfitting based on decision theory (Minin et al., 2003).
MRBAYES was run for 10
For the CO I gene, a total of 38 haplotypes were found among the 46
sequenced
Bayesian phylogeny of
Phylogenetic analysis of the combined data set revealed distinct geographic
groups generally supported by high posterior probabilities (Fig. 2). We
named these groups according to their geographical origin. Distinct groups
were found in the western, southern and eastern parts of the Alps, whereas
no specific group was located in the central parts of the Alps. Populations
analyzed from outside the Alps were both strongly different (e.g., Corsica)
but also very similar (e.g., Great Britain) from the populations of the Alps.
Bayesian trees calculated across the individual genes (Figs. S3 and S4 in the Supplement)
revealed a very similar grouping of at least the basic lineages when
compared with the Bayesian tree of the combined data set (Fig. 2). The
relationships among these lineages, however, differed considerably between
the data sets. All basal splits were only weakly supported by posterior
probabilities. In an attempt to increase the support for the basal branches,
we included the secondary structures of the 18S rRNA gene (for methods, see
Additional Methods S5). However, also with this approach, the basal splits
were only weakly supported. Although we sequenced only one individual from
each population, we found a positive correlation between geographic
distances (log
Relationship between pairwise genetic distances of the COI gene
(patristic distances from tree shown in Fig. S3) and pairwise geographic
distances of
The main results of our study can be summarized as follows: we found
(i) considerable genetic diversity within
The distinct lineages recognized in the present study may represent
different species. In general, species are considered cryptic if they have
been erroneously classified as a single species because of a lack of
distinct morphological features. DNA barcoding has revealed cryptic
diversity in a variety of taxa (Nieberding et al., 2005; Bickford et al.,
2006; Witt et al., 2006; Lefébure et al., 2007; Pauls et al., 2010).
Lefébure et al. (2006b) showed that clades of crustaceans diverging
by more than 0.16 substitutions per site of the CO I gene as measured by
patristic distances are likely to present distinct species. Based on the
threshold distance suggested by Lefébure et al. (2006b) a minimum of 10
distinct species of
Pfenninger and Schwenk (2007) showed that cryptic species are evenly
distributed among the major metazoan taxa. This suggests that a simple body
plan is not a sufficient explanation for an increased occurrence of cryptic
species within a taxonomic group. Some authors (e.g., Bickford, 2006) have
suggested a link between extreme environmental conditions and cryptic
species. Extreme environments impose stabilizing selection on changes in
morphology during speciation, resulting in morphological stasis (Rothschild
and Mancinelli, 2001; Nevo, 2001; Lefébure et al., 2006a; Bickford,
2006; Lefébure et al., 2007).
Given that we propose the existence of cryptic species and since our
sampling scheme is based on one individual per site only, we cannot detect
multiple species at one location. In fact, it is surprising that we still
found moderate spatial structuring in our data. According to our
phylogenetic analysis of
Our study provides some evidence that the orogenic history of the alps and
presumably the climatic variability during the Quaternary promoted genetic
diversity of
Sequences are available at
The authors declare that they have no conflict of interest. Edited by: Jutta Stadler Reviewed by: two anonymous referees